Previous experiments purportedly showed that image-based factors like convexity were sufficient for figure assignment. Recently, however, we found that the probability of perceiving a figure on the convex side of a central border was only slightly higher than chance for two-region displays and increased with the number of display regions; this increase was observed only when the concave regions were homogeneously colored. These convex figure context effects (CEs) revealed that figure assignment in these classic displays entails more than a response to local convexity. A Bayesian observer replicated the convex figure CEs using both a convexity object prior and a new, homogeneous background prior and made the novel prediction that the classic displays in which both the convex and concave regions were homogeneous were ambiguous during perceptual organization.Here, we report three experiments investigating the proposed ambiguity and examining how the convex figure CEs unfold over time with an emphasis on whether they entail recurrent processing. Displays were shown for 100 ms followed by pattern masks after ISIs of 0, 50, or 100 ms. The masking conditions were designed to add noise to recurrent processing and therefore to delay the outcome of processes in which they play a role. In Exp. 1, participants viewed two- and eight-region displays with homogeneous convex regions . In Exp. 2, participants viewed putatively unambiguous hetero-convex displays. In Exp. 3, displays and masks were presented to different eyes, thereby delaying mask interference in the thalamus for up to 100 ms.The results of Exps. 1 and 2 are consistent with the interpretation that recurrent processing is involved in generating the convex figure CEs and resolving the ambiguity of homo-convex displays. The results of Exp. 3 suggested that corticofugal recurrent processing is involved in resolving the ambiguity of homo-convex displays and that cortico-cortical recurrent processes play a role in generating convex figure CEs and these two types of recurrent processes operate in parallel. Our results add to evidence that perceptual organization evolves dynamically and reveal that stimuli that seem unambiguous can be ambiguous during perceptual organization. A central function of perception is segregating the visual field into foreground objects and their local backgrounds, yet the underlying mechanisms are not fully understood. Foreground-background perception  was long thought to result from low-level processes in a feedforward perceptual system. Evidence for this view was provided by demonstrations that figure assignment was determined by image-based cues such as convexity. For example, for stimuli like the one on the right in What processes produce these global CEs? The lack of convex figure CEs for We are particularly interested in whether feedback from higher to lower levels in the visual hierarchy  plays a role in convex figure CEs and in resolving ambiguity during perceptual organization. It is reasonable to assume that the homogeneous background prior entails perceptual completion, which seems to require feedback  participated in Exp. 1B. The number of participants whose data were removed because they did not meet the standard criterion was eight for Exp. 1A, four for the follow-up experiments to Exp. 1A, and eight for Exp. 1B.homo-convex displays (56 per region number condition) comprising alternating low luminance  and high luminance  convex and concave regions  that filled the screen (17.7°H x 22.8°W) of a 21-in Sony CRT monitor. The HL and LL regions were equal luminance steps below and above the backdrop; hence, contrast with the backdrop did not serve as a depth cue  was presented 0, 50, or 100 ms after the experimental display. homo-convex test display appeared for 100 ms. Participants' task was to report whether the red probe on the display was located “on” or “off” the region they perceived as the figure shaped by the nearest border. This probe on/off task provides a valid and reliable index of figure assignment near a border  and display-to-mask ISI condition. Participants in Exp. 1B viewed 96 trial-unique homo-convex displays. Participants made their on/off judgment regarding the red probe by pressing the top or bottom button on a custom button box. Assignment of buttons to “on”/“off” responses was balanced across subjects. Before the experimental trials, participants completed eight practice trials; none of the displays used in the practice trials appeared in the experimental trials. Participants were left alone to complete the experimental trials.On experimental trials in Exp. 1A, each participant viewed 56 randomly presented trial unique The proportion of trials on which the convex region closest to fixation was perceived as the figure/object was calculated for each participant by summing the number of trials on which they reported “on” when the probe appeared on the convex region, and “off” when the probe appeared on the concave region and dividing this sum by the total number of trials on which they responded .F = 19.39, p < 0.001, η2 = 0.094 and with display-to-mask ISI, F = 4.98, p < 0.009; η2 = 0.051. Importantly, an interaction between region number and ISI, F = 3.06, p < 0.05; η2 = 0.032, showed that convex figure reports increased with ISI for eight-region displays, F = 4.894, p =0.01, η2 = 0.095, but not for two-region displays, F < 1. To represent the magnitude of the convex figure CEs, the difference between convex figure reports for eight- vs. two-region displays was calculated for each ISI condition. This CE index was not statistically different from zero in the 0-ms ISI condition ; it just reached statistical significance in the 50-ms ISI condition  and was robust in the 100-ms ISI condition . The CE index was statistically higher in the 100-ms than the 50-ms display-to-mask ISI condition, p < 0.002  = 33.219, p < 0.001, η2 = 0.0174, but there was no effect of ISI, F = 2.002, p > 0.13  X 3 (ISI) ANOVA. A significant main effect of region number was observed, 0.13 see .Together with the follow-up experiments, the results of Exp 1 show that convex figure CEs reached asymptote for 100-ms displays in the 100-ms display-to-mask ISI condition−200 ms after stimulus onset. It is plausible that pattern masks shown 0 and 50 ms after the offset of a 100-ms stimulus (and maybe longer up to 100 ms) interfere with recurrent processing following the initial analysis of the test display, thereby preventing the emergence of convex figure CEs. We continue to investigate the feasibility of this interpretation in subsequent experiments reported in this article. At this point, an explanation holding that the test display-off signal is critical for convex figure CEs and that masks interfere with that signal in the 0-ms display-to-mask ISI condition remains possible  = 13.354, p < 0.001, η2 = 0.125. Neither a main effect of display width, F = 2.197, p = 0.140, η2 = 0.01, nor an interaction between display width and display-to-mask ISI was observed, F < 1.0. The finding that convex figure CEs for narrow and wide displays showed the same developmental trajectory over variations in display-to-mask ISI characterizes the recurrent processes as operating between levels of the visual hierarchy rather than within a level .To better characterize recurrent processes implicated by the results of Exp. 1A, we compared convex figure reports obtained for narrow eight-region displays in the three display-to-mask ISI conditions to those obtained for the wider eight-region displays in Exp. 1A. The gray bars in homo-convex displays in the 50-ms display-to-mask ISI condition and larger convex figure CEs in the 100-ms ISI condition. That masks shown up to 100 ms after stimulus offset interfered with the generation of convex figure CEs is consistent with the hypothesis that recurrent processes play a role. Recall, however, that it has been proposed that homo-convex displays are ambiguous because when convex regions are homogeneous the convexity object prior and the homogeneous background prior oppose each other. If this proposal is correct, Exp. 1 may have assessed the need for recurrent processing in ambiguity resolution as well as in convex figure CEs.In Exp. 1, we found statistically significant convex figure CEs for hetero-convex displays. Hetero-convex displays are unambiguous because disconnected heterogeneous convex regions are unlikely to be completed into a single surface  were tested in Exp. 2. When they entered the laboratory, they were assigned via an ABBA order to one of two display-to-mask ISI conditions: 0 or 50 ms. The data from two participants were excluded from the analysis because they did not meet our response rate criterion. The data from one additional participant were excluded because they pressed the response button immediately after pressing the foot pedal. A total of 42 participants (28 F) took part in a follow-up experiment in which displays were exposed for 80 ms and were followed immediately by a 200-ms mask. They were assigned via an ABBA procedure to view either the same eight-region displays viewed by participants in Exp. 2 or 56 two-region displays used in Experiment 1A.hetero-convex regions alternated with homo-concave regions; these were the experimental stimuli. In the other half, hetero-convex regions alternated with hetero-concave regions; these were filler stimuli included to reduce tendencies to form a strategy of always reporting either the homo or the hetero regions as figures  and varied in width, subtending a mean visual angle of 13.59° (range: 11.54–15.65°). The convex regions differed in color. The convex region sharing the central border with the concave region was always gray. The other convex regions were filled with one of four colors: yellow, magenta, cyan, or orange. These colors appeared once per display, and across displays appeared on each of the remaining three convex regions equally often. The concave regions were filled with either HL or LL gray. The convex and concave regions differed in contrast polarity: when the concave regions were HL, the convex regions were LL and vice versa. Samples are shown in In the filler displays, the alternating regions were HL or LL and colored gray, yellow, magenta, or cyan. The two central regions were always filled with HL and LL gray; hence, the central regions in the two types of displays were equated. The remaining colors were used to fill the other regions. The same color was never used to fill two consecutive regions; nor was it used in multiple convex (or concave) regions in a single display. Convex regions were HL in half the displays and LL in the rest. The convex and concave regions differed in contrast polarity: when the luminance of the concave regions was high, that of convex regions was low and vice versa. Michelson contrast at the central border = 0.72. Michelson contrasts at the other borders ranged from 0.62 to 0.78.The mask that followed the figure-ground display consisted of a geometric pattern that measured 6.0° H x 17.7° W . The ANOVA showed a main effect of region number, F = 45.838, P < 0.001, η2 = 0.270 and an interaction between region number and ISI, F = 5.077, p = 0.026, η2 = 0.039. Convex figures reports for eight-region displays increased with display-to-mask ISI (as in Exp. 1), whereas convex figure reports for two-region displays did not. The results of Exp. 2 are consistent with the interpretation that convex figure CEs entail recurrent processes.The results obtained with eight-region F = 10.738, p = 0.002, η2 = 0.148 . This finding is inconsistent with a claim that the absence of convex figure CEs for homo-convex displays in the 0-ms display-to-mask ISI condition of Exp. 1 can be explained by mask-induced interference with a display offset signal as per Macknik and Martinez-Conde  <1; the CE index was 0.04]. Together, the results of Exp. 2 and this follow-up experiment suggest that, when object and background priors are not in opposition for convex regions, the processes that produce convex figure CEs in eight-region displays take more than 80 ms after display onset and continue for up to 150 ms . These findings accord with previous estimates of how long perceptual completion takes although our displays are different from those examined by previous authors  than for homo-convex displays (Exp. 1)? We have attributed this temporal difference to processes that resolve the ambiguity of homo-convex displays. This raises the question of whether ambiguity resolution occurs in parallel with the generation of alternative interpretations for homo-convex displays or whether it occurs in a later decision process. It is reasonable to assume that perceptual completion processes generating background interpretations for homogeneous regions are underway while homo-convex displays are exposed as well as while hetero-convex displays are exposed, yet convex figure CEs are evident in convex-figure responses 50 ms later for homo- than for hetero-convex displays. We found that the cost of ambiguity resolution is approximately constant with increases in the ISI between eight-region displays and the subsequent backward masks: AN ANOVA comparing convex figure reports for eight region displays in the 0- and 50-ms ISI conditions common to both Exps. 1A and 2 showed a main effect of display type, F = 15.681, p < 0.001, η2 = 0.112 (higher convex figure reports for hetero- than homo-convex displays); and a main effect of ISI, F = 5.11, P = 0.026, η2 = 0.04 (higher convex figure reports in the 50-ms ISI condition than the 0-ms ISI condition). No interaction between display type and ISI was observed, F = 1.273. This analysis reveals that the disadvantage for convex figure reports in eight-region homo- vs. hetero-convex displays is present in the 0-ms condition and remains stable as convex figure CEs develop. This result is consistent with the interpretation that ambiguity resolution processes operate in parallel with the processes generating convex figure CEs. Indeed, evidence for convex figure CEs in homo-convex displays lagged behind evidence for convex figure CEs in hetero-convex displays by ~50 ms: Convex figure reports for eight-region homo-convex displays in the 50-ms ISI condition where convex figure CEs first emerged in Exp. 2  were statistically equivalent to convex figure reports for eight-region hetero-convex displays in the 0-ms ISI condition where convex figure CEs first emerged in response in Exp. 2 , p > 0.29. Similarly, convex figure reports for eight-region homo-convex displays in the 100-ms ISI condition of Exp. 1A  were statistically indistinguishable from convex figure reports for eight region hetero-convex displays in the 50-ms ISI condition in Exp. 2 , p > 0.29. These results suggest that resolving the ambiguity of homo-convex displays adds ~50 ms to the time at which CEs are evident in convex figure reports.Why do convex figure CEs emerge earlier in time for We have interpreted the evidence presented so far as consistent with the proposal that convex figure CEs entail recurrent processes. In Exp. 3, we used dichoptic presentations to investigate whether the relevant recurrent processes extend to the thalamus or whether they operate solely within the cortex. In dichoptic presentations, test displays and masks are presented to different eyes, as illustrated in homo- and hetero-convex displays and masks to different groups of observers under dichoptic presentation conditions and compared the results to the results obtained for eight-region displays in Exps. 1 and 2, respectively. Only eight-region displays were used because, in previous experiments, convex figure CEs were evident in increased convex figure reports with increases in display-to-mask ISI for eight-region displays but not for two-region displays  participants viewed eight-region hetero-convex displays intermixed with filler displays and their masks under dichoptic masking conditions. Data from 23 participants did not meet our response rate criterion; eliminating their data from the analysis left 32 participants in each of the 0, 50, and 100-ms display-to-mask ISI conditions for each display type. Assignment to ISI condition was random.A total of 215 undergraduate students  from the University of Arizona participated in Exp. 3. Of these subjects, 113  viewed eight-region http://www.psy.vanderbilt.edu/faculty/blake/Stereoscope/stereoscope.html). Experimental displays and masks were shown on the left and right monitor locations equally often and, hence, were presented to the left and right eyes equally often.A haploscope was used with a head and chin rest to present the stimuli dichoptically. In the haploscope, a pair of mirrors reflected to the left and right eyes images that were reflected to them by a second set of mirrors aimed at locations on the left and right sides of a monitor, such that each of these monitor locations were visible to one eye only (see homo- and hetero-convex displays and filler displays were created in a size visible in the haploscope mirrors (6.45°H X 10.06°W). For each set, masks were created by cropping the masks used in Experiments 1 and 2. The masks measured 7.11°H X 12.42°W.New sets of eight-region Participants were seated 51.3 cm from the monitor, with distance controlled by a chinrest. Before the experimental trials, the mirrors of the haploscope were adjusted for each participant individually until the left and right-eye images of a nonius fixation cross were aligned. This procedure assured that images presented on the left and right side of the screen were aligned and centered on the fixation cross.In each trial the test display and the mask were presented to different eyes. In half of the trials the display was presented to the left eye and the mask to the right eye; in the other half of the trials (randomly intermixed), the display was presented to the right eye and the mask to the left eye. Participants were unaware that the images were presented to different eyes.homo-convex eight-region displays made their figure reports by pressing one of two vertically aligned buttons on each trial to indicate whether they perceived the black or white regions as figures. For the hetero-convex displays participants reported whether an elongated rectangular probe  centered vertically in either a convex or concave region to the left or right of the central edge appeared to be “on” or “off” the figure (as in Exps. 1 and 2). Peterson and Salvagio .For hetero-convex displays equally often in Exps. 2 (monoptic presentations) and 3 (dichoptic presentations) in the 0-ms and 50-ms ISI conditions. A between-experiment ANOVA showed that, for hetero-convex displays, convex figure reports increased as ISI increased from 0 to 50 ms, F = 10.506, p < 0.002, η2 = 0.078, replicating Exp. 2 . Neither a main effect of presentation type nor an interaction between presentation type and ISI was observed for hetero-convex displays, Fs <1. The absence of an effect of presentation type is consistent with the interpretation that cortico-cortical recurrent processes are involved in generating the convex figure reports in eight-region hetero-convex displays. This interpretation is not surprising inasmuch as evidence suggests that convexity is represented in cortical area V4  than in Exp. 1B (monoptic presentations) in the 0- and 50-ms display-to-mask ISI conditions, but not in the 100-ms ISI condition where previous experiments indicated convex figure CEs for masked homo-convex displays had reached asymptote. This pattern was shown to be statistically significant by main effects of presentation type (Exp) and ISI, F = 39.86, p < 0.001, η2 = 0.24 and F = 12.23, p < 0.002, η2 = 0.09, respectively. An interaction between presentation type and ISI was also observed, F = 4.37, p < 0.04, η2 = 0.034: Convex figure reports for homo-convex displays were statistically higher with dichoptic presentations than with monoptic presentations in both the 0-ms and 50-ms ISI conditions (ps < 0.008) but not the 100-ms ISI condition, p > 0.06, where previous evidence suggested that convex figure CEs for 100-ms displays reached asymptote. This finding suggests that cortico-thalamic feedback occurring up to 50 ms after stimulus offset plays a role in resolving the ambiguity of homo-convex displays. When interference from the mask in subcortical areas was removed for a period of time by presenting the mask to a different eye than the experimental display in Exp. 3, ambiguity resolution proceeded without interference and convex CEs reached asymptote in the 50-ms ISI condition, 50 ms earlier than when monoptic presentations of the display and mask were used in Exp. 1.As can be seen in homo- and hetero-convex displays obtained with dichoptic presentation conditions in Exp. 3 and found that they were equivalent (see homo- vs. hetero-convex) and ISI  revealed a main effect of ISI, F = 10.03, p < 0.001, η2 = 0.097 but not a main effect of Display Type, F = 0.497, p = 0.482, nor an interaction between Display Type and ISI, F = 0.434, p = 0.648. The finding that with dichoptic presentations the convex figure CEs emerge and reach asymptote for homo- and hetero-convex displays in the same display-to-mask ISI conditions suggests that cortical-thalamic recurrent processes involved in ambiguity resolution occur in parallel with cortico-cortical recurrent processes producing convex figure CEs. If ambiguity resolution occurred later, convex figure reports would reach asymptote in a longer ISI condition for homo- than hetero-convex displays even under dichoptic presentation conditions.We next compared convex figure reports for lent see . A 2 X 3homo- and hetero-convex displays. This finding contrasts with what was found with monoptic presentations of the experimental display and its mask , where convex figure CEs emerged later in time for homo-convex than for hetero-convex displays. We attributed the additional time to ambiguity resolution, suggested by Goldreich and Peterson's . Thus, it is likely that high levels of the visual hierarchy are engaged in the iterative cortico-thalamic activity that plays a role in resolving the ambiguity of homo-convex displays. Indeed, Sillito and Jones . Third, as mentioned previously, differential difficulty of figure-ground decisions made in high levels cannot account for the differences between hetero- and homo-convex displays observed in Exps. 1 and 2 because those differences are not evident in Exp. 3.Can the results of our experiments be due to the disruption of feedforward activity in high levels of the visual hierarchy rather than to the disruption of recurrent processes? We consider that unlikely for the following reasons: First, the earlier emergence of convex figure CEs for homo- and hetero-convex displays? The convex and concave regions in the former displays differ in luminance only, whereas those in the latter displays differ in both color and luminance. There is some evidence that stimuli defined by luminance differences only are processed differently from those defined by both luminance and color differences. For instance, Rivest and Cavanagh .It would be interesting to use a within-subjects design to test the questions addressed here and to include more fine-grained manipulation of ISI. It would be difficult, although not impossible, to present trial-unique displays in a within-subjects experiment, so the conditions would necessarily be somewhat different. Although we did not report the results in the body of the paper, we did test intermediate ISIs of 25 and 75 ms for homo-convex displays with dichoptic presentation conditions and found the results fell between the results obtained for the adjacent ISI conditions.homo-convex displays and suggest that cortico-cortical recurrent processes play a role in generating convex figure CEs.The three experiments presented here are consistent with the interpretation that recurrent cortico-thalamic processes are involved in resolving the ambiguity of eight-region The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by Human Subjects Protection Program UArizona. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.MP conceptualized the experiments, performed statistical analyses, engaged in theoretical discussions, and wrote the paper. EC created the stimuli, wrote the programs, conducted the experiments, performed statistical analysis, and engaged in theoretical discussions. All authors contributed to the article and approved the submitted version.


According to the ideomotor principle, repeated experience with an action and its perceivable consequences (effects) establish action-effect associations. Research on verbal instructions indicates that such associations are also acquired from verbal information. In the present experiment (N = 651), first, we aimed to replicate unintentional response-priming effects from verbal action-effect instructions . Second, we investigated the involvement of perceptual processes in the verbally induced response-priming effect by perceptually presenting (Condition 1) versus not presenting (Condition 2) the color that was subsequently named as an effect in the instructions. Third, we tested a saliency-based explanation of the verbally induced response-priming effect by highlighting all components (action and effect) without an association between them (Condition 3). Overall, we found the predicted response-priming effect following verbal action-effect instructions . Condition 2, which did not include perceptual information in the instructions, still showed a significant response-priming effect but was descriptively weaker compared to the effect of the replication Condition 1. Condition 3, which merely highlighted the action and effect component without endorsing an association, did not show a significant effect. In sum, our study provides further solid evidence that verbal instructions lead to unintentional response-priming effects. Other conclusions must be considered preliminary: The between-condition comparisons were descriptively in the predicted direction—perceptual aspects are relevant, and a saliency-based account can be excluded—but the differences in accuracy between conditions were not statistically significant. According to the ideomotor principle, goal-directed actions are driven by anticipatory representations of their effects , responses are facilitated . When the associated response is different from the required response , responses are impeded .We use the term “compatibility effects”  when resThere is ample evidence for compatibility effects resulting from action-effect learning based on direct experiences ;. HoweveAnother study  showed aThe experimental procedure in these two recent publications on verbal action-effect instructions  is similupright or italic by pressing the left or right key. Thus, instruction combinations for the diagnostic task were either compatible or incompatible with the action-effect instructions presented for the inducer task. The results showed a compatibility effect in the diagnostic task, pointing to an effect of the instructions on the subsequent performance.For example, Theeuwes et al.  providedDamanskyy et al.  also proTwo questions arise from this previous research. First, Damanskyy et al.  involvedIn the present research, we investigated three central aspects related to the effect of verbally induced action-effect instructions. First, we sought to provide a high-powered replication of an unintentional response-priming effect from a verbally processed – but never directly executed – action-effect contingency. Second, we investigated the relevance of perceptual processes for verbal action-effect learning. Third, we tested the idea that verbal action-effect instructions establish associative links against a saliency-based alternative explanation. The experimental procedure of this study was similar to Damanskyy et al. . In the Our study consisted of three between-participant conditions. The first condition  served as a standard for comparing the remaining two conditions and is an exact replication of Damanskyy et al. . In thisThe second condition  was identical to the first, except the blue color was not presented before the verbal instructions. This was designed to address the role of perceptual aspects in the link-formation process. If the presentation of the actual color before the verbal instructions influences the compatibility effect, this suggests that the learning mechanism that mediates the effect between reading the verbal instruction and observing a response-compatibility effect does not rely solely on language-like symbolic processes. Instead, perceptual processes would contribute to that mechanism.The third condition  was designed to test the associative-learning account against a saliency-based alternative explanation. To that aim, the instruction presentation did not include the verbal action-effect contingency, but instead presented the effect and the action components independently from each other. On one page of the task instructions participants were informed that pressing a specific key  was important and they should thus memorize the statement ‘I will press the [right/left] key.’ Later, on a different instruction page, participants were presented with the blue color sample and were informed that this color was relevant and will appear during the categorization task. In other words, we highlighted both components  but did not facilitate an association between them. If previous findings i.e.,  and thosIn sum, to provide further information about the mechanism involved in verbal action-effect learning, we compared three conditions. The “visual-verbal link” condition facilitated associative learning and provided exact information about the perceptual properties of the perceptual component. The “verbal link only” condition also facilitated associative learning between the instructed perceptual and action component but did not include the exact perceptual properties. The “no verbal link” condition highlighted the perceptual and action components but did not facilitate associative learning between them. We expected the strongest response-compatibility effect in the visual-verbal link condition, replicating Damanskyy et al. . We expeM = 32.1, SD = 8.8). The participants were recruited by the online participant recruitment platform Prolific and received monetary compensation. We removed four participants who participated in the study twice due to technical errors. The study was approved by the local ethics committee, and all participants provided informed consent. The required sample size to find the central response-compatibility effect was not calculated prior to the analysis but was instead based on prior experiences with a similar design . Participants’ age ranged from 18 to 50  followed by the action-effect instructions. In the verbal link only condition, participants saw only the action-effect instructions. In the no verbal link condition, participants were presented separately with instructions for a specific response and an example of the to-be-presented color. The data collection of the no verbal link condition reported in this manuscript was done after the data collection of the other two conditions, once we realized that a design error  in the originally-collected third condition made it impossible to calculate a comparable response-compatibility effect. Because of this error and the subsequent changes, the relationship between the present research and the initial pre-registration (https://osf.io/qfmc6) is complicated. However, the overall hypotheses and technical details of the analyses  remain the same. Analyses were conducted only after data collection was completed for all conditions.The study included three main conditions: required response and effect prime) and one between-subject factor (instructed response). Required response represented a factor that specified what response was required from participants according to the categorization task instructions . Effect prime specified whether the blue screen was present  or absent (control) in a given trial. Instructed response was a between-participant factor indicating the instructed response in the action-effect instructions  or response instructions . Key assignment to vowels/consonants was counterbalanced between participants.All three conditions included two within-subject factors . They were also informed that this instruction was important and they were instructed to memorize the sentence. After some intermediate instructions, the participants were presented with the color blue and told that this color will appear in the subsequent categorization task. A few minutes passed between memorizing the critical action-effect instructions and starting the test-phase task. These minutes where filled with action-effect unrelated instructions .In the visual-verbal link condition, we presented an example of the critical stimulus (the color blue) and told them that this would be the color referred to later in the instructions. Afterwards, participants were presented with the action-effect instructions: e.g., “The categorization task was identical for all conditions. The presented stimulus was either a vowel  or a consonant , and each appeared an equal number of times in random order. During this part, the participants judged whether a presented stimulus was a vowel by pressing the left key (A) or a consonant by pressing the right key (L). Along with each presented letter, the background color was either blue  or gray . All stimulus-response combinations were equally distributed between the effect-prime present and effect-prime absent trials. We implemented a response deadline of 1500 ms. If a response was incorrect or longer than 1500 ms, an error feedback message was displayed for 1500 ms. Participants performed eight practice trials and 96 test trials. The practice trials did not include any critical trials . Instructions for the test phase included information that the background color may change during the task and they were explicitly instructed to ignore these color changes and focus on the vowel-consonant categorization task.ez package; We used the R software package to prepare and analyze the data . Response errors and reaction times were analyzed with a mixed ANOVA (Responses with missed deadline were omitted (0.83%). Prior to the response time analysis, we removed all error responses (3.43%). No response was faster than 150 ms . In addition, we removed trials with response times beyond the mean +/– 3 times the standard deviation calculated by participant and within-participant conditions (1.11%).We applied ANOVA with Type II Sums of Squares for statistical analysis as recommended for unbalanced groups and for models in which an interaction effect is of interest . FurtherF = 14.1, p < .001, ηp2 = .02. The 4-way interaction effect including the three main conditions did not reach the conventional significance level, F = 2.08, p = .149, ηp2 < .01. The non-significant trend could indicate an effect in the opposite direction than predicted: for example, showing no replication of Damanskyy et al.  = 10.08, p = .002, ηp2 = .04. We analyzed the experimental effect within prime present and prime absent trials separately. The expected 2-way interaction effect between required response and effect prime was significant for the trials with the effect prime present F = 7.36, p = .007, ηp2 = .02. The same 2-way interaction effect was marginally significant for the trials with the effect prime absent F = 3.31, p = .070, ηp2 = .01. The visual inspection of both 2-way interactions  = 3.98, p = .047, ηp2 = .01. We analyzed the data further separately for the prime present and prime absent trials. For the critical prime-present trials, the 2-way interaction effect was not significant F = 1.89, p = .171, ηp2 = .00. Similarly, the same interaction effect was also not significant for prime-absent control trials F = 2.41, p = .122, ηp2 = .01. Visual inspection of the result pattern  = 1.13, p = .289, ηp2 = .00. The separate analyses for the prime present F = 2.07, p = .152, ηp2 = .00 and prime absent trials F = 0.24, p = .622, ηp2 = .00 also did not show significant effects. In contrast to the previous two conditions, the 3-way interaction effect between required response, effect prime and instructed response was not significant As the magnitude of each compatibility effect is not easily visible from the three 3-way interaction effects illustrated in p = .149), the result pattern is descriptively in the predicted direction. The visual-verbal link condition shows the strongest compatibility effect, and the effect in the verbal link only condition is weaker. The no verbal link condition shows the weakest compatibility effect.As illustrated in F = 1.24, p = .266, ηp2 = .00. The 4-way interaction effect including the three main conditions was also not significant F = 0.23, p = .635, ηp2 = .00. To stay consistent with the response errors analysis and to evaluate potential speed-accuracy trade-offs, we analyzed each condition separately. None of the 3-way or 2-way interaction effects are significant . Appendix C presents the full ANOVA result tables for the reaction times analysis.The 3-way interaction effect between required response, effect prime, and instructed response was not significant F = 1.03, p = .310, ηp2 < .01. The analysis revealed a significant main effect of prime, indicating that participants responded slower on critical trials than on control trials. We analyzed further the experimental effect within effect prime present  and effect prime absent (control) trials separately to evaluate potential influence of speed accuracy trade-off. The 2-way interaction between required response and effect prime in critical trials was not significant F = 0.05, p = .831, ηp2 < .01. The same 2-way interaction was also not significant within control trials F = 1.61, p = .206, ηp2 < .01. These results indicate the speed-accuracy trade-off did not affect participants’ response errors.The 3-way interaction between required response, prime, and instruction response was not significant F = 0.49, p = .483, ηp2 < .01. We analyzed further the experimental effect within effect prime present  and effect prime absent (control) trials separately to evaluate the potential influence of a speed-accuracy trade-off. The 2-way interaction was not significant in both critical trials F = 0.09, p = .765, ηp2 < .01 and control trials F = 0.48, p = .487, ηp2 < .01.The 3-way interaction between required response, prime, and instructed response was not significant F = 0.12, p = .735, ηp2 < .01. We analyzed further the experimental effect within effect prime present  and effect prime absent (control) trials separately to evaluate the potential influence of a speed-accuracy trade-off. The 2-way interaction was not significant in both critical trials F = 1.88, p = .172, ηp2 < .01 and control trials F = 2.30, p = .131, ηp2 < .01.The 3-way interaction between required response, prime, and instructed response was not significant The compatibility scores of response times were calculated in the same ways as the compatibility scores of response errors.The aim of the present study was to provide a further test of verbally induced response-compatibility effects and to investigate some of the underlying conditions. We will start by discussing the visual-verbal ink condition as it is an exact replication of previous results that we included as a baseline against which to compare the outcomes of the other conditions.The results from the visual-verbal link condition replicated findings to the study of Damanskyy et al. . Based oThe verbal link only condition aimed to evaluate the role of a perceptual component in the effect of verbal instructions on action. In this condition participants did not see the example of the blue color patch before processing the action-effect instructions. The analysis still revealed a significant compatibility effect in the expected direction. As in the visual-verbal link condition, participants made fewer errors in prime-present trials when the required response matched the instructed response from the action-effect instructions, and the response time analyses did not indicate a speed-accuracy trade-off. By itself, this condition that includes only a small design change provides another replication of unintentional response-priming effects from verbal instructions.Descriptively, the compatibility effect in this condition was smaller than in the visual-verbal link condition see  and WolfThe no verbal link condition was included to evaluate whether the effect of action-effect instructions is based on associative learning, or if the results could be explained by a saliency-based alternative account. If the compatibility effect indeed stems from an associative link between the stimulus and response components and not merely familiarity with these components and an independent temporary activation of both components, then presenting them separately from each other  should result in a weaker (or absent) compatibility effect. For the first time in a series of five tests , there iThe central limitation of the present research is in the non-significant 4-way interaction effect. Thus, while we have evidence that the instructions affected subsequent responses (3-way interaction effect), our conclusions related to differences between the conditions should be considered in relation to related research and as a starting point to continuously putting them to the test. We nonetheless presented the descriptive condition differences as they corresponded to our hypotheses. We expected the strongest compatibility effect for the visual-verbal link condition, and a comparatively weaker effect in the verbal link only condition. Furthermore, we expected and found the weakest compatibility effect in the no verbal link condition.Overall, the present findings are based on the analysis of response errors. The analysis of response times serves to rule out a speed-accuracy trade-off as an explanation of the findings, mirroring the pattern found by Damanskyy et al. . Mekawi The descriptive pattern of response errors and response times shown in The present study provides another illustration of a verbally-induced response-compatibility effect. Unintentional response-priming effects are well documented e.g., , but typBeyond this central effect, we provide some initial evidence that perceptual aspects play a role in the mechanism that mediates the effect of verbal information on subsequent responses, and we find additional support for an associative-learning mechanism from verbal information to action by providing evidence against an explanation based solely on familiarity  of the individual perception and action components. These findings are in line with previously-presented theoretical perspectives , 2017 thhttps://osf.io/28m6u/?view_only=fb3e8821628e41f1bf688ae71118b873.The datasets generated during the current study are available via the Open Science Framework (OSF): https://osf.io/28m6u/?view_only=fb3e8821628e41f1bf688ae71118b873.The R script and R output are also available vis OSF: https://osf.io/qfmc6.The pre-registered hypothesis is also available via OSF: The additional file for this article can be found as follows:10.5334/joc.284.s1Appendices.Appendice A, B to C.


Imaging wide-field surface plasmon resonance (SPR) microscopy sensors based on polyacrylic acid polyelectrolyte brushes (PAA PEBs) were designed to enhance the sensitivity of nano-object detection. The switching behavior of the PAA PEBs against changes in the pH values was investigated by analyzing the chemical, morphological, optical, and electrical properties. At pH ~1, the brushes collapse on the surface with the dominance of carboxylic groups ( Polyelectrolyte brushes (PEBs) are charged poly acids or bases grafted to the substrate surface which undergo reversible protonation or deprotonation upon shifting the pH value across the acidity of the brush ,2. This The physical binding between the polyelectrolyte brushes with the macromolecules remains an open question. For instance, several researchers have stated that the proteins bind to the PEBs even when they have the same net charge , which cPolyacrylic acid (PAA) has gained attention due to the existence of a carboxylic group on each repeating unit that changes its charges, structures, and hydrophilicity due to the variation in pH ,12. Incral state .Recently, imaging wide-field SPR microscopy sensors (WF-SPRM) have gained substantial attention in the real-time detection of viruses and virus-like particles due to their capability to measure low particle concentrations with a high sensitivity ,15,16. IVarious studies have been conducted to enhance the sensitivity and selectivity of the SPR sensors . G. del This work describes the design of the WF-SPRM based on PAA PEBs deposited on a Au layer, which improves the sensitivity of the SPR sensing device. Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies were employed to investigate the transformation of the carboxylic acid  or sodium hydroxide (NaOH) to the final solution. Adding HCl leads to a reduction in pH through the delivery of 3, SF10 glass, n = 1.72) with a 5 nm titanium adhesion layer and, subsequently, with an approximately 41-45 nm gold layer via a magnetron sputtering technique . These gold substrates were further cut and cleaned using piranha solution (v/v) for 10 min. Afterward, ITO, silicon, and the gold substrates were rinsed with water and dried at ambient conditions.Before surface functionalization, indium-tin-oxide (ITO) glass substrates (100 nm ITO on glass) and n-type silicon wafers (0.5 mm-thick) were cleaned by sonicating in acetone and isopropyl alcohol for 30 min under ambient conditions. In addition, the gold substrates were prepared by first coating a glass slide  and near-ambient-pressure X-ray photoelectron spectroscopy (NAP-XPS). 1H NMR measurements were conducted at 600.13 MHz using a 5 mm high-resolution multi-channel and broadband (5 mm BBO model) equipped with Z-gradient.The chemical structure was investigated using The swelling behavior and the dissociation degree were investigated using FTIR (Bruker VERTEX 80/80v Vacuum FTIR Spectrometers) and NMR (Bruker AVANCE III NMR spectrometer). In addition, surface morphology and wettability were studied using an atomic force microscope (SPM SmartSPM™-1000) and water contact angle measurements (WCA) for a water droplet (pH = 7) of size 10 μL taken on three occasions. These measurements were performed at room temperature for PAA-SH PEBs at different pH values .The optical transmittance and reflectance spectra were measured using a double-beam UV–Vis spectrophotometer (Hitachi U-3900H) with a total integration sphere. After that, the optical constants  layer is based on Kretschmann’s scheme of plasmon excitation ,30. The lsewhere ,31. The n = 1.59 ) with an1H-NMR spectrum of PAA PEBs shows signals at 1.1, 1.2–1.7, and 2.01 ppm ascribed to The main challenge in using PAA PEBs as a coating on metal films or other substrates is the lack of a functional group that strongly binds to the surface. Therefore, a fraction of carboxylic groups of PAA were modified with thiol functional groups binding to a gold layer. PAA-SH was synthesized using carbodiimide amide coupling reactions between PAA and cysteamine with specific molar ratios to obtain a modified one in four carboxylic acid groups a 25]. T. T25]. TA matrix b 33,34],341H-NMRA matrix c–f. Casa (−COO−) c. XPS O (O=C−O−) d. In addeV (N−C) e. The XPmeasured f. These −1  . At pH =function b 36]. T. TCOOH) terature ,38.1H-NMR spectra of PAA PEBs for different pH values are shown in In addition, To better understand the effects of the dissociation of the carboxylic acid groups of PAA PEBs on the structure following pH variation, schematic diagrams in addition to 2D AFM measurements of PAA PEBs at different pH values  are shown in COOH) a. This soughness b. The ca brushes c. The doic state  13]. Th. ThCOOH)Increasing the solvent pH increases the electrical conductivity of the PAA PEBs, which can be attributed to the sh layer . In addish layer . This resh layer . Figure l factor . The actt pH = 5 b, which f 685 nm c. Upon t−1].WF-SPRM was employed to record a series of 2D images demonstrating local spatiotemporal changes in intensity, which helps to detect the binding of nano-particles. Thiolated PAA (PAA-SH) was deposited onto the gold sensor surface using the spin coating method a. Figure3)3 complex composite film as a coating for the Au layer to enhance the sensitivity of the WF-SPRM. We found that the S/N for Au-(PANI-HCl)/Al(NO3)3 with S/N of 10 ± 1. Therefore, we can conclude that coating the Au layer with PAA brushes is more efficient than our previous work. This can be attributed to the fact that the PAA brushes have a higher refractive index than the (PANI-HCl)/Al(NO3)3 complex composite film.Our work aims to describe the influence of the coating gold sensing surface with the PAA-PEBs on the sensitivity of the WF-SPRM sensor, as well as to provide characteristics of the PAA-PEBs. For this, the pH-dependent switching properties of the PAA-PEBs and their effects on the optical and electrical characterizations were studied. It is demonstrated that, at pH ~1, the initial state of the PEBs corresponds to the brushes that collapse to the surface, with the dominance of the carboxylic groups (


Respiratory viruses can cause epidemics or pandemics, which are worldwide outbreaks of disease. The severity of these events varies depending on the virus, its characteristics, along with environmental factors. The frequency of epidemics and pandemics caused by respiratory viruses is difficult to predict, but the potential severity of such events underlines the importance of continued monitoring, research, and preparation for emerging infectious diseases. To help improve pandemic preparedness, we created a fully integrated duplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) device targeting two respiratory viruses, influenza A/X-31 virus and bovine coronavirus, as a replacement for SARS-CoV-2. This device can be adapted to any other respiratory virus. In this study, we showed and evaluated a prototype of a microfluidic system, and showed that duplex RT-LAMP can detect and distinguish between the two viruses, with LoDs of 2,000 copies/ml for bovine coronavirus and 200 copies/ml for influenza A/X-31 virus. Respiratory viruses have the potential to cause epidemics or pandemics, which are widespread outbreaks of disease  remains the gold standard for respiratory virus detection, and there are examples of multiplex panels detecting different respiratory viruses by RT-PCR  to demonstrate the feasibility of duplex LAMP on a lateral flow test (LFT) 2) to integrate all the steps needed for LAMP diagnostics on a chip and to make the test easy to perform without laboratory infrastructure 3) to evaluate the performance of an integrated duplex LAMP on a chip.Essential features of PoC tests are the rapid detection of their specific targets and their device-independency. Here, we designed an instrument-free, stand-alone prototype for the detection of two respiratory viruses – influenza A/X-31 virus (InfA) and bovine coronavirus (BovCov) as a BSL-2 laboratory-feasible replacement for SARS-CoV-2.The microfluidic structures on the chip allow sample loading, RT-LAMP reaction, and subsequent LFT detection of the amplification product. The chips were produced in several iterations by 3D-printing of the embossing forms, and subsequent hot embossing of the chip molds Fig. .Fig.1MicThere are three main parts integrated in the microfluidic chip design. First, a 50 µl reaction chamber containing the lyophilized reagents for RT-LAMP (colored with phenol red for better observation of fluidic processes) and an inlet for sample loading. Second is a cavity with an outlet containing an LFT for the detection of amplified LAMP products. Third, a blister filled with water for the lateral flow assay. All parts are connected to the reaction chamber by a microfluidic channel system.Bst 2.0 polymerase) e.g., on a heating plate or similar heating system. Subsequently, the blister is pressed flushing the reaction mixture through the microfluidic channel to the LFT, initiating lateral flow detection. After 20–30 min, the LFT is ready for read-out.The workflow can be summarized as follows: an aqueous sample is added into the inlet of the chip, flowing to the reaction chamber by capillary forces, thereby resolving the lyophilized LAMP reagents. The RT-LAMP reaction can then be performed at a temperature between 60–72 °C  for both RT-LAMP reactions was therefore determined using purified RNA with known copy numbers. tenfold dilution series of RNA were tested with the duplex RT-LAMP device. The RNA dilutions were tested separately due to the competing behavior of the BovCov LAMP reaction mentioned previously. Additionally tenfold dilution series were tested with quantitative RT-LAMP using the LightCycler 480 system  as a reference.The replicates of 1.000 copies and 100 copies of BovCov RNA were positive in quantitative RT-LAMP after an incubation time between 15–18 min Fig. A. The reThe same samples were tested with the duplex RT-LAMP device. For BovCov RT-LAMP, a LoD of 100 RNA copies  was determined with three positive replicates showing intense test bands Fig. A, confirFor InfA RT-LAMP (consistent with quantitative RT-LAMP), a LoD of 10 RNA copies  was determined with three positive replicates, two showing a moderate test band and one showing a weak test band Fig. A. For 5 The most widely used PoC tests for respiratory viruses are rapid antigen tests that detect viral proteins. These tests are affordable, simple to use and give quick results, but can have low sensitivity and hence present lower negative predictive values than nucleic-acid amplification assays , and a reliable activation of lateral flow test . We have developed a chip design that enables these functionalities without instrumental fluidic control, and, moreover, without active valves or other active mechanical elements on the chip. This system concept enables its cost-saving single-piece manufacturing and makes it a promising diagnostic tool especially in the areas where the pricing for diagnostic devices is a particularly limiting factor.We have also looked into other published multiplex isothermal amplification methods integrated on microfluidic platforms. Most of them present similar limits of detection, and some of the studies showed tests with capabilities for detecting two or more targets . N-gene of bovine coronavirus (European Virus Archive Ref-number: 022 V-04370) and M-gene of influenza A/X-31 virus (GenBank number: DQ874879.1) were chosen as target sequences. The sequences of the LAMP primer sets are listed in Table For lateral flow assay, LF primer of the bovine coronavirus (BovCov) primer set was labeled at the 5′ end with biotin and LB primer with 6-carboxyfluorescein (FAM). LF primer of the influenza A/X-31 (InfA) primer set was labeled with FAM and LB primer with digoxigenin. Primers were synthesized by Eurofins Genomics Germany GmbH .Bovine coronavirus was obtained from European Virus Archive . Influenza A/X-31 virus was obtained from the Robert Koch-Institute . Virus RNA was isolated from frozen cell culture supernatant using Monarch Total RNA Miniprep Kit . Copy numbers of isolated RNA were determined using two-step reverse transcription droplet digital PCR (data not shown).RT-LAMP reagents for a 50 µl reaction volume were freeze-dried in the reaction chamber of the chip. The RT-LAMP mix contained 12.5 µl of lyophilization-friendly WarmStart LAMP 4X Master Mix that includes reverse transcriptase , 5 µl of each of the 10X primer mixes for BovCov and InfA , 12.5 µg BSA , 6% (v/v) trehalose , and 0.1 mM phenol red . Phenol red was added for the visualization and monitoring of fluidic processes on the chip and played no functional role in LAMP reaction.The lyophilization was performed in a freeze-dryer Christ Epsilon 2–6 . After lyophilization, a LFT with two detection lines, one binding to biotin and the other to digoxigenin  was placed in the chip, and the bottom surface of the chip was sealed with ThermalSeal RTS sealing foil . A blister filled with 350 µl double-distilled water  was mounted over the dedicated area using an O-shaped piece of a double-sided sticky tape. The chips were stored at 4 °C until use.Bst 2.0 polymerase as stated by NEB.Lyophilized reagents in the chips were resolved by adding 65 µl of a sample. Samples consisted of the respective RNA dilution in PCR-grade water  and 0.1% (v/v) Triton X-100  to reduce surface tension, thereby facilitating microfluidic flow. Absolute copy numbers were calculated for a volume of 50 µl, representing the total volume of the reaction chamber. Inlet and outlet were sealed with gas permeable adhesive Air-O-Seal . The LAMP reaction was performed for 30 min on an in-house-built electricity-free heating system producing a consistent temperature between 72 °C and 60 °C (unpublished data), optimal for Subsequently, LFT-based detection of the LAMP product was initiated by pressing the water-filled blister until the reaction mixture reached the strip. The result of the LFT was ready for read-out after 20–30 min and stable for several days.


Leucobacter chromiiresistens, the most closely related species in terms of nomenclature. However, a digital DNA–DNA hybridization analysis between these two species revealed a maximum identity similarity of only 27.5%. We found phenotypical and genomic differences between strain Marseille-Q4368 and its closely related species. These findings underscore the classification of this bacterium as a distinct species. Hence, we propose the name Leucobacter manosquensis sp. nov. strain Marseille-Q4368 (=CSUR Q4368 = DSM 112403) for this newly identified bacterial species.Extending our knowledge on human skin microbiota is a challenge to better decipher its role in health and disease. Using the culturomics method, we isolated strain Marseille-Q4368 from the healthy forehead of a 59-year-old woman. We describe here the main characteristics of this bacterium using a taxonogenomic approach. This new bacterial species is Gram-positive, non-motile, and non-spore-forming. Its 16S rRNA sequence exhibited a similarity of 99.59% with  Pharmaceutical and cosmetic innovation have long been based on the search for innovative compounds, often extracted from plants. A major turning point in the history of medicine, with a strong impact on human health, came with the discovery of antibiotics and their natural producers: microorganisms. Fungal and bacterial microorganisms represent promising sources of new compounds for prophylactic or therapeutic treatments. While the medical applications arising from our knowledge on digestive microbiota are now widely used and recognized, they are not yet as well developed concerning cutaneous microbiota and the potential beneficial impact of pre-probiotics and bacteriotherapy on the skin. Not-yet-cultured microorganisms are certainly an underestimated source of interesting compounds. Extending our knowledge on human skin microbiota is a challenge to better decipher its role in health and disease. Recent studies have shown that human skin harbors a wide diversity of microbes, far beyond commonly isolated bacteria. Many of the bacteria that are part of the skin microbiota are difficult to isolate because of difficulties in reproducing the human skin environment in vitro . KnowledLeucobacter genus belongs to the Microbacteriaceae family and was first proposed in 1996 by Takeuchi et al., with the first species named Leucobacter komagatae [Leucobacter takes its name from the morphology of the cells, which are rods and light-colored. Currently, there are 41 known species, of which 33 have been validly published with a correct name . Moreover, the species found within the genus have been isolated from different environments and Leucobacter manosquensis is the first species of this genus to have been isolated from healthy human skin.Since 2012, our laboratory has set up an innovative approach aiming at surpassing the limitations associated with culture-independent techniques. These new strategies, called culturomics and taxonogenomics , consistLeucobacter manosquensis, a new bacterial species isolated from the healthy skin using a taxonogenomic polyphasic approach. It includes phylogenomic analyses, wall fatty acid composition, and phenotypic characterization.Here, we describe 2 area of skin from the forehead and hands of volunteer healthy women. The samples were collected using sterile swabs soaked in the Culture Top® transport medium . The study was validated by the ethics committee Sud-Est IV (reference ID-RCB: 2019-A01508-49). Each sample mixed with the transport media was serially diluted, and 50 µL of each dilution was incubated under aerobic conditions at 31 °C after being seeded (DS) in Columbia Agar . To identify the strains, a Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry protein analysis was carried out in triplicate using a Microflex spectrometer . Whole genome sequencing (WGS) was performed as described below. The strain spectra were imported into the MALDI BioTyper software  and analyzed using standard pattern matching with the default parameters. Our database  was then incremented with the spectra of this new bacterial species.Samples were obtained by swabbing a 10 cmDifferent growth temperatures , atmosphere conditions  using generator bags , and pH conditions  were tested. The biochemical properties of these strains were tested using API ZYM, API 20 NE, and API 50 CH strips  in accordance with the manufacturer’s instructions. To evaluate the bacterial structure, a colony was collected from agar and immersed into a 2.5% glutaraldehyde fixative solution. The suspension was vortexed, passed ten times through a 21-gauge needle to separate the bacterial colonies, and fixed on an uncoated glass slide using cytocentrifugation. A 1% ammonium molybdate negative stain was applied for 1 min before gently washing the slide with 0.2 µm filtered distilled water. The slide was air-dried and examined using scanning electron microscopy on a TM4000 microscope  with a 15 kV voltage. A motility test was performed using the semi-solid TTC media.Cellular fatty acid methyl esters (FAME) analysis was performed using GC/MS. Two samples of each strain were prepared with approximately 110 mg of bacterial biomass per tube harvested from several culture plates. The fatty acid methyl esters were prepared as described by Sasser (2006) . The spohttps://www.ncbi.nlm.nih.gov/refseq/, accessed on 4 September 2023).The genomic DNA (gDNA) of the strain Marseille-Q4368 was extracted in two steps: a mechanical treatment was first performed with glass beads being acid-washed  using a FastPrep-24™ 5G grinder  at maximum speed (6.5) for 90 s. Then, after 30 min of lysozyme incubation at 37 °C, the DNA was extracted using the EZ1 biorobot  with the EZ1 DNA tissue kit. The elution volume was of 50 µL. The gDNA was quantified using a Qubit assay with a high-sensitivity kit  to 0.2 ng/µL. The genomic DNA was then sequenced using the MiSeq technology  with the paired end strategy prepared with the Nextera XT DNA sample prep kit . To prepare the paired end library, dilution was performed to require 1 ng of each genome as input. The “tagmentation” step fragmented and tagged the DNA. Then, limited-cycle PCR amplification (12 cycles) completed the tag adapters and introduced dual-index bar codes. After purification with AMPure XP beads , the libraries were then normalized to specific beads according to the Nextera XT protocol (Illumina). The normalized libraries were pooled into a single library for sequencing on the MiSeq. The pooled single-strand library was loaded onto the reagent cartridge and then onto the instrument, along with the flow cell. To improve the quality of the assemblies, an Oxford Nanopore approach was performed on 1D genomic DNA sequencing for the MinIon device using the SQK-LSK109 kit. The library was constructed from 1 µg of genomic DNA without fragmentation and end repair. Adapters were ligated to both ends of the genomic DNA. After purification with the AMPure XP beads , the library was quantified using a Qubit assay with the high-sensitivity kit . For bacterial identification, we used the “What’s in my pot” (WIMP) workflow , which uses the RefSeq sequence database from the NCBI , esterase lipase (C8), leucine arylamidase, cystine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase, D-mannitol, D-glucose, D-maltose, malic acid, and α-mannosidase. However, the remaining reactions yielded negative results. Additionally, the catalase test was positive, while the oxidase test was negative. The differential phenotypical characteristics of The genome size of strain Marseille-Q4368 was 3,184,602 bp long with a 67.34% G + C content. The genome assembly of this strain was achieved on three contigs (with 9.44× coverage). Of the 2976 predicted genes, 2870 were protein-coding genes and 59 were RNAs  . Leucobacter manosquensis showed a coherent structure compared to its closely related species  of proteins show that the genome of  species .L. japonicus and L. musarum at 95.3%. On the other hand, the lowest value was shared between L. japonicus and L. aridicollis at 75.1%. Consequently, strain Marseille-Q4368 had the highest OrthoANI value of 84.3% with L. chromiiresistens, which appears to be the closest phylogenetic species . Additionally, the tRNA molecules were more numerous than the rRNA and other RNA molecules, ranging between 46, 47, and 48 depending on the Leucobacter species analyzed. Furthermore, the difference in the G + C percentage between the species was very low, mostly being less than 4%. This reinforces our thesis that the Marseille-Q4368 strain is a new species within the Leucobacter genus. More statistical details from the genomes are showed in In terms of genome size, the strain Marseille-Q4368 (3.18 Mbp) had approximately a similar genomic size compared to all the genomes of the Leucobacter manosquensis . Strain Marseille-Q4368 exhibited facultative aerobic growth, with a growth rate of 31 °C within 24 h. This particular bacterium was Gram-positive and had non-motile, non-spore-forming bacterial cells that were rod-shaped, measuring approximately 1.4 × 0.3 µm. 12-methyl-tetradecanoic acid (69%), 14-methyl-hexadecanoic acid (16%), and 14-methyl-pentadecanoic acid (7%) were the main branched fatty acids retrieved in strain Marseille-Q4368. Esculin ferric citrate, esterase (C4), esterase lipase (C8), leucine arylamidase, cystine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase, D-mannitol, D-glucose, D-maltose, malic acid, and α-mannosidase were the present enzymes. It tested positive for a catalase test and negative for an oxidase test.T was 3.18 Mbp with 67.3 mol% of G + C content.The genome of strain Marseille-Q4368The genome and 16S rRNA sequence were deposited in GenBank under accession numbers MW583464 and JAFEVO000000000, respectively.T (=CSUR Q4368 = DSM 112403) was isolated from the forehead of a 59-year-old woman with healthy skin.The type strain Marseille-Q4368The skin is the largest organ of the human body, and because of its external nature, skin disorders have been the focus of medical research and cosmetic trials since ancient times. Having evolved together, scientific knowledge and cosmetic formulations are now inseparable. It should be noted that cosmetic and pharmaceutical regulations are particularly demanding, and are constantly updated in light of new scientific advances. The skin is colonized by a large number of organisms with which it evolves in symbiosis: the cutaneous microbiota. The number of symbiotic microorganisms is greater than the number of skin cells. Although it is not (yet) possible under actual European regulations to add living microorganisms to a formulation, growing knowledge on this subject already offers clear new prospects. Thus, the discovery of new bacteria could be a starting point for innovating component findings. Leucobacter manosquensis was discovered from a healthy skin swab sample. Both phylogenetic and phenotypic analyses revealed several different characteristics of the strain Marseille-Q4368 when compared to other members of the genus Leucobacter, which belongs to the Microbacteriaceae family and the Actinobacteria phylum and was first proposed in 1996 . Therefo


The OST-ME had a droplet size of 16.18 ± 0.02 nm and a low polydispersity index (0.09 ± 0.00). In vitro drug release from OST-ME fitted well to the Higuchi release kinetics model. Cytotoxicity assays demonstrated that OST-ME was not notably toxic to human corneal epithelial cells (HCECs), and the formulation had no irritation to rabbit eyes. Ocular pharmacokinetics studies showed that the areas under the concentration–time curves (AUC0-t) in the cornea and conjunctiva were 19.74 and 63.96 μg/g*min after the administration of OST-ME, both of which were 28.2- and 102.34-fold higher than those after the administration of OST suspension (OST-Susp). Moreover, OST-ME (0.1%) presented a similar therapeutic effect to commercially available dexamethasone eye drops (0.025%) on CNV in mouse models. In conclusion, the optimized OST-ME exhibited good tolerance and enhanced 28.2- and 102.34-fold bioavailability in the cornea and conjunctiva tissues compared with suspensions in rabbit eyes. The OST-ME is a potential ocular drug delivery for anti-CNV.Osthole (OST), a natural coumarin compound, has shown a significant inhibitory effect on corneal neovascularization (CNV). But, its effect on treating CNV is restricted by its water insolubility. To overcome this limitation, an OST-loaded microemulsion (OST-ME) was created to improve the drug’s therapeutic effect on CNV after topical administration. The OST-ME formulation comprised Capryol-90 (CP-90), Cremophor The cornea is highly specialized avascular tissue , a featuCnidium monnieri (L.) Cusson and has been verified to have potential therapeutic applications due to its multiple significant pharmacological activities, including anticancer, antiosteoporotic and antiproliferative effects. Previous studies have also shown that OST not only reduces intraocular pressure . H. H21]. H0–t value of the corneal and conjunctival tissue in the OST-ME group were 19.74 and 63.96 μg/g·min, respectively. Therefore, the ME displayed a notable advantage over OST suspension in increasing the permeability of OST across the cornea and conjunctiva.The distributions of OST in the corneal and conjunctival tissue are shown in p < 0.05) (p > 0.05). These results demonstrated the OST-ME can restrain CNV.The anti-CNV effect of OST-ME was evaluated. As shown in  < 0.05) B, but thThe H&E section was used to evaluate the structural difference among every group. As illustrated in p < 0.05) after administration of the corresponding drug. The result illustrated that OST-ME could restrain the level of VEGF-A and MMP-9 in the CNV model , which illustrated that OST release from OST-ME was dependent on Fickian diffusion [In vitro drug release has a significant effect on predicting and understanding the nature of a formulation. The drug release curve of the OST-ME and OST-Susp were better fitted to the Higuchi and first-order equations, and the Riffusion . AlthougWhen ophthalmic drug delivery systems for topical application are developed, ocular safety is always a critical issue. The Draize eye test is a commonly used way to assess the irritation of eye drops and it was used to assess the irritation caused by OST-ME in this study . Here, tThe pharmacokinetic study was conducted in rabbit eyes. This study had strengths and weaknesses and could directly evaluate the drug concentration in the corneal and conjunctiva tissue. However, since cornea and conjunctiva tissue are not consecutively sampled, it is impossible to harvest tissues from the same animal at every time point. So, the drug level will be impacted by individual absorption and metabolism to some extent . CompareVarious causes can result in CNV, but chemical injury is clinically one of the most common. In this experiment, an alkali burn injury model was used to induce corneal angiogenesis and evaluate the treatment effect of OST-ME. The results demonstrated that the OST-ME (H) treatment could significantly inhibit CNV when the eye drop was instilled four times a day. The antiangiogenic effects of OST-ME were verified by evaluating the area of CNV B and H&EOsthole (OST), isopropyl myristate (IPM), and monoolein were provided by Macklin Biochemical Co., Ltd. . Medium-chain triglycerides (MCTs) were purchased from Yunhong Chemical Preparation Excipients Technology Co., Ltd. . Ethyl oleate and triacetin were obtained from J&K Scientific Technology Co., Ltd. . Oleic acid was provided by Tokyo Chemical Industries Co., Ltd. . Propylene glycol dicaprylate (PGD) and castor oil were provided by Hunan Er-Kang Pharmaceutical Co., Ltd. . PEG400 was obtained from Solarbio Life Science . EL-35 was obtained from Shanghai Puzhen Biotechnology Co., Ltd. . Kolliphor HS-15 (HS-15) was obtained from BASF SE . Tween-80 (TW-80) was obtained from Sichuan Jinshan Pharmaceutical Co., Ltd. . TSP and CP-90 were acquired from Gattefosse . Glycerin was provided by Huikang Pharmaceutical Co., Ltd. . Sodium hyaluronate (SH) was obtained from Furuida Biochemical Co., Ltd. . A Cell Counting Kit-8 (CCK-8) was acquired from APExBIO . Mice MMP-9 and VEGF-A enzyme linked immunosorbent assay (ELISA) kits were acquired from Elabscience Biotechnology . Dexamethasone (DEX) sodium phosphate solution were provided by Huaqing Pharmaceutical Co., Ltd. .New Zealand white rabbits (2.0–2.5 kg) and male BALB/c mice  were obtained from Huaxing Experimental Animal Breeding Co. . All rabbits and mice were provided a standard daily diet and water and placed in lab that was kept at 23 °C ± 2 °C with a 12 h light/12 h dark cycle. Every animal was experimented on according to the principle of the Association for Research in Vision and Ophthalmology (ARVO) declaration.® C18 column , and the column temperature was set at 40 °C. The mobile phase was made up of methanol–water  under an isocratic conditions flow rate of 1.0 mL/min and the injection volume was 10 μL. The ultraviolet (UV) detector wavelength was 322 nm. The OST concentration was determined from the standard curve.The OST concentration was measured by the high performance liquid chromatography (HPLC) method according to a previous report ,42. BrieTo obtain suitable ingredients for the preparation of OST-MEs, the solubilities of OST in different oils , surfactants  and cosurfactants (PEG400 and TSP) were investigated according to a previous report ,44. All The ability of the selected surfactant to emulsify the oil phase was assessed according to a previous report . EmulsioThe aforementioned three ingredients  were chosen to constitute the PTPDs by the water titration phase inversion emulsification (PIE) method according to a previous report . The are® software . CCD-RSM was applied to investigate the effects of independent variables  and their interaction over the dependent variables  [The OST-ME formulation was optimized by the experimental statistical design technique with the Design ExpertY2: PDI) ,49. The w/v) SH solution was added to the OST-ME solution, and the total volume of the mixed solution was 20 mL. The OST-ME was filtered using a 0.22 µm filter. Each sample was prepared in triplicate.The optimal OST-ME formulation could be obtained from the CCD-RSM optimization results and was prepared using the PIE method. Briefly, three selected ingredients were weighed and homogenously mixed at 37 °C. Then, OST was added in the homogenous mixture and dissolved under magnetic stirring. Then, distilled water was added in the mixture until the transparent light blue solution was obtained. Lastly, 10 mL of 0.2% (v/v) with purified water, and each experiment was performed in triplicate.The average DS, ZP and PDI values of the OST-ME were tested by dynamic light scattering . All samples were diluted  to separate the free OST from the OST-ME by centrifuge . All samples were assayed by the aforementioned HPLC method. The value was calculated by the following equations:The morphology of the optimized OST-ME was observed by transmission electron microscopy (TEM) . Briefly, OST-ME was diluted 50-fold with purified water before the TEM study. Then, the diluted OST-ME was applied to a carbon-coated copper grid. After drying, OST-ME samples were observed and photographed by TEM at 25 °C.The pH and osmolality of the OST-ME were tested by pH meter and freezing point osmometer . Each experiment was conducted in triplicate.−1 [In this study, the OST, Blank-ME, OST-ME and the physical mixture (PM) of 20 mL of Blank-ME/OST  were characterized by an AVATAR 370 FTIR instrument . All samples were measured at 25 °C in the range of 4000–400 cm−1 .The stability of the optimized OST-ME was assessed for three months. Briefly, the OST-ME was prepared by filtration sterilization, and five milliliters of the sample was loaded in sterilized polypropylene eye drop bottles, sealed with a cover under a cleaning bench and then placed at 4 °C and 25 °C. Both the physical and chemical stabilities of OST-ME formulations were evaluated. All tests were performed on day 0 and at 1, 2 and 3 months, and samples were analyzed in triplicate.The drug release of OST was conducted via dialysis in simulated tear fluid , containing 0.1% TW-80 as the solubilizer, which served as the release medium. The release of OST was analyzed by HPLC, and the cumulative release percentage (Q) of OST was calculated at relevant time points (according to Equation (3)). The Q was fitted to the zero-order kinetics, first-order kinetics, Higuchi and Korsmeyer–Peppas models, and the release curves were drawn using Origin software . The OST-Susp was chosen as the control.0 is the total weight of the OST in the OST-ME, Cn is the OST concentration in STF at tn, V is the total volume of STF, Vi and Ci are the sample volume and concentration at ti, respectively, and tn is the nth sampling time [Here, Wing time .4 cells per well were exposed to different concentrations of OST-ME (5 μg/mL and 10 μg/mL) and corresponding concentrations of blank-ME, which were cultured for 0.25, 1, 2 and 4 h. Then, 10% CCK-8 (100 μL) was added to every well and cultured for 4 h, and the optical density (OD) of all samples was tested at 450 nm with a microplate reader . All results were calculated by Equation (4) [The toxicity of OST-ME to HCECs was evaluated with CCK-8 assays. HCECs at a density of 1 × 10tion (4) .(6)CellThe irritation of the OST-ME was measured by six white rabbits according to the Draize eye test ,54. NoneForty-two healthy rabbits were randomly divided into two groups , which were randomly divided into seven subgroups (n = 3), respectively. Both eyes of each animal were instilled with 50 μL of 0.1% OST-ME in the test group and the OST-Susp in the control group. The animals were euthanized by an injection of 4% phenobarbital sodium solution at scheduled time points. The corneal and conjunctival tissues were collected. Then, the tissues were weighed and stored at −80 °C for HPLC analysis. The drug in the tissue samples was extracted according to a previous report .A validated method reported in a previous study was revised to gain OST from corneal and conjunctival tissue . Brieflyw/v), OST-ME , OST-ME  or dexamethasone . Each group was treated four times a day for a total of 7 days.The CNV mouse model was established using the alkali burn method described in a previous report . After oAfter treatment days 1, 3 and 7, CNV images of all animals were taken by microscope . To quanl radius .After day 7, three mice were sacrificed and the whole eyeball was enucleated from each. The corneas were separated and fixed in FAS trimming for hematoxylin-eosin (H&E) staining and histopathological observations.The expression levels of VEGF-A and MMP-9 in the corneas were measured. After days 3 and 7, five mice were sacrificed in each group, and the corneal samples were dissected and saved at −80 °C until analysis. The corneal samples were rewarmed at 4 °C for half an hour before analysis. The samples were treated according to the previous method . The conp < 0.05 was considered as statistical significance. All data were recorded as the mean ± standard deviation (SD).All results were analyzed by SPSS software . Fisher’s least significant difference (LSD) test was recorded to compare the differences between every group; In this study, an optimized OST-ME formulation was successfully generated and characterized. The formulation showed good tolerance. Moreover, the formulation had good storage stability at 4 °C. In vitro drug release studies illustrated that OST-ME had a better sustained drug release rate than OST-Susp. In vivo pharmacokinetic studies illustrated that OST-ME had better bioavailability than OST-Susp and that the retention time of OST-ME on the cornea was prolonged. In addition, OST-ME effectively inhibited CNV and decreased VEGF-A and MMP-9 protein expression. In summary, OST-ME could be a potential drug for anti-CNV.


When t/d decreased from 8.6 to 2.4, σb decreased from 1458 MPa to 1132 MPa, σs decreased from 917 MPa to 730 MPa, and the elongation decreased from 0.31 to 0.28 at ultra-low temperatures. When t/d decreased from 8.6 to 2.4, the surface roughness of the specimen increased from 0.769 to 0.890 at room temperature and increased from 0.648 to 0.809 at ultra-low temperatures. During the microplastic deformation process of Ni-Co-based superalloy thin sheets, the coupled effects of surface roughening caused by free surface grains and hindered dislocation movement induced by grain boundary resulted in strain localization, which caused fracture failure of Ni-Co-based superalloy thin sheets.With the development of product miniaturization in aerospace, the nuclear industry, and other fields, Ni-Co-based superalloys with excellent overall properties have become key materials for micro components in these fields. In the microforming field, size effects significantly impact the mechanical properties and plastic deformation behavior of materials. In this paper, micro-tensile experiments at room temperature and an ultra-low temperature were carried out to study the effects of initial microstructure and deformation temperature on the deformation behavior of Ni-Co-based superalloy thin sheets. The results show that as the ratio of specimen thickness to grain size ( Product miniaturization is one of the main trends in advanced manufacturing technology. Micro-electro-mechanical systems (MEMS) emerged under this situation. Microforming technology has the characteristics of large volumes, high efficiency, high precision, low costs, and no pollution and has become a key technology for the manufacturing of thin sheet microstructure parts ,2,3. Supt/d, and a “smaller is weaker” size effect appeared. In the microforming of metal thin sheets, the strain coordination ability between grains decreased with decreasing t/d, resulting in a decrease in the homogeneous plastic deformation ability. Nie et al. [t/d. As t/d decreased, the fracture mechanism changed from ductile fracture to brittle fracture. Zhao et al. [t/d for pure copper foils with thicknesses ranging from 100 to 600 μm.To name a few, previous studies ,22,23,24e et al.  conducteo et al.  conducteo et al.  conducteo et al.  conducteo et al. ,30 foundExisting research shows that ultra-low temperatures play a very good strengthening and toughening role in face-centered cubic (FCC) metal ,32,33. TTo date, research on size effects mainly focuses on room temperature and high temperatures. Ultra-low temperature forming is still a relatively new and potential forming method to improve the mechanical properties of metal materials. The deformation characteristics of materials at ultra-low temperatures are very different from those at room temperature, such as mechanical properties, microstructure evolution, fracture mechanism, etc. Therefore, this study carried out micro-tensile tests on Ni-Co-based superalloys at room temperature and an ultra-low temperature. The microstructural evolution behavior during micro-tensile deformation was characterized and analyzed using electron backscatter diffraction (EBSD) and scanning electron microscopy (SEM). The impacts of grain and ultra-low temperatures on the micro-tensile properties and deformation mechanism of Ni-Co-based superalloy thin sheets are clarified. This study provides a theoretical basis for the forming process of Ni-Co-based superalloy thin sheet micro components and has practical application value in promoting its application in the microforming field.2H5OH+20 mL HCl+3 gCuSO4, and the metallographic structure was observed using an OLYMPUS microscope .Cold-rolled and annealed Ni-Co-based superalloy thin sheets were selected as the experimental material. The main mass percentages of Ni-Co-based superalloys were 25% Co, 10% Cr, 4% Mo, 1% W, 2% Al, 4.5% Ti, 1% Nb, and the rest was Ni. To obtain specimens with different initial grain sizes, solution heat treatment schemes were designed, namely 1150 °C/4 h, 1150 °C/6 h, 1150 °C/8 h, 1200 °C/2 h, 1200 °C/4 h followed by water quenching (WQ). WQ was to maintain the microstructure at high temperatures. To prevent Ni-Co-based superalloy from oxidation during the heat treatment process, the vacuum heat treatment method was used. The solution specimen was ground with 400–1000 grit SiC metallographic water sandpaper. The specimen was mechanically polished using a diamond metallographic spray polishing agent with a particle size of 0.5~2.5 μm until there were no obvious scratches on the surface of the specimen. Chemical etching was carried out in a mixed solution of 20 mL C−1. The ultra-low temperature tensile device included a cryogenic chamber, a temperature control device, and a liquid nitrogen tank, as shown in 2H5OH+10% perchloric acid. A scanning step size of 0.5 μm and a binning of 4 × 4 were used for EBSD  characterization. The fracture morphology of the specimens was characterized and analyzed using SEM .The dog-bone-shaped micro-tensile specimens with a gauge width of 2 mm and a gauge length of 6 mm were prepared along the rolling direction (RD) using electrical discharge machining (EDM). A 30 kN electronic universal testing machine  was used to conduct the micro-tensile test, and the strain rate was set to 0.01 sα represents the proportion of grains in the surface layer of the specimen; t represents the thickness of the specimen (μm); W represents the width of the specimen (μm); d represents the average grain size of the specimen (μm).At the mesoscopic scale, materials no longer behave as isotropic strain-hardening materials. The surface layer model is currently a widely recognized theory in the microforming field ,45. The α was 25.1%. However, when the grain size was 84.8 μm, α reached 86.1%, which led to an obvious softening effect of flow stress.t/d. When t/d decreased from 8.6 to 2.4, the tensile strength σb decreased from 1221 MPa to 1090 MPa, and the yield strength σs decreased from 793 MPa to 622 MPa at room temperature. When t/d decreased from 8.6 to 2.4, σb decreased from 1458 MPa to 1132 MPa, and σs decreased from 917 MPa to 730 MPa at ultra-low temperatures. The engineering stress of the micro-tensile specimen with a grain size of 84.8 μm increased with increasing engineering strain. However, the increasing degree of engineering stress was slightly lower compared to those of micro-tensile specimens with other grain sizes. This was because the surface layer grain deformation was less constrained due to the existence of the free surface, and dislocations were not easily accumulated in the surface layer grains, resulting in a reduction in the strain hardening ability of the surface layer grains and lower deformation resistance. At the same t/d, the stress of the specimen increased significantly with decreasing deformation temperature. It could be inferred that the ultra-low temperature was beneficial in improving the strength of the materials. The elongation of the alloy was better due to the matrix γ phase of the face-centered cubic (FCC) structure. When t/d decreased from 8.6 to 2.4, the elongation decreased from 0.26 to 0.21 at room temperature, and the elongation decreased from 0.31 to 0.28 at ultra-low temperatures. As the proportion of the surface layer grains increased, the number of grains contained in the thickness direction decreased, weakening the deformation coordination ability. Thus, the elongation of the Ni-Co-based superalloy decreased with decreasing t/d. At the same t/d, the elongation of the specimen increased significantly with decreasing deformation temperature. In summary, the strength and elongation of materials under the ultra-low temperature conditions were significantly better than those at room temperature. Therefore, ultra-low temperatures had a strengthening and plasticizing effect on the Ni-Co-based superalloys.The engineering stress–strain curve has universal significance because the curve reflects various deformation processes such as plasticity, yield, and fracture that occur in the material under the action of external forces. Plastic strains can be characterized by local micro-orientations in the material . KAM repAs shown in When the grain size was small, the number of grains in the thickness direction of the specimen was large, and the deformation of the grains could be distributed to adjacent grains to coordinate the deformation, making the plastic deformation relatively homogeneous. As the grain size increased, the number of grains in the thickness direction of the specimen decreased, and the proportion of surface layer grains increased, which produced significant size effects. Therefore, as the proportion of the surface layer grains increased, the deformation coordination ability of materials weakened during the micro-tensile deformation process. At the same time, soft-oriented grains were easy to deform, while hard-oriented grains were difficult to deform. The material tended to follow the direction of minimum acting. The reduction in grains contained in the thickness direction of the thin sheet tended to form a “weak and weak” bond, causing strain concentration in the specimen and premature fracture.As the deformation temperature increased, the KAM value of the Ni-Co-based superalloy specimen decreased, and the deformation ability decreased. When the strain was 0.19, obvious concentrated areas with larger KAM values appeared in the surface grains and internal grains of the specimen at room temperature. However, the areas with larger KAM values were more evenly distributed throughout the grain at ultra-low temperatures. The KAM value distribution of the fractured specimen in The plastic deformation and ductile fracture at the mesoscopic scale are very different from those at the macroscopic scale. Surface roughening occurs during the plastic deformation of polycrystalline materials, which affects the subsequent plastic deformation behavior and formability of the material. The evolution of specimen surface roughness is mainly affected by the initial microstructure and deformation conditions. To explore the effects of grain size and deformation temperature on material flow behavior during micro-tensile deformation, the surface roughness was measured using a confocal laser scanning microscope (CLSM)  on the gauge length part of the specimen with a scanning step of 0.3 μm and an observation area of 640 μm × 640 μm.Differences in crystal orientation between adjacent grains led to surface roughening at the mesoscopic scale . PolycryAdditionally, the surface roughness of specimens at ultra-low temperatures is lower than that at room temperature. This may be due to the fact that an ultra-low temperature reduces the dislocation activation energy and inhibits dislocation recovery, which increases the dislocation density within the grain. Compared with the deformation at room temperature, the deformation at ultra-low temperatures is borne more within the grain, thereby reducing the overall rotation of the grain. Thus, the deformation becomes more homogeneous, resulting in less surface roughening.η of the specimen decreased from 0.902 to 0.816, with a decrease rate of 9.5%. The increase in the proportion of the surface layer grains promoted the development of local necking and reduced the deformation uniformity.As shown in η decreased from 0.920 to 0.889 with a decrease rate of 3.4%. The difference in deformation between the surface grains and the internal grains was reduced, indicating that the specimen deformed more homogeneously at ultra-low temperatures. High local misorientation was mainly concentrated inside the grains at ultra-low temperatures, and the storage capacity of dislocations was enhanced. The weakening of strain concentration at grain boundaries resulted in a delayed onset of cracks. Thus, ultra-low temperatures enhanced the ductility of Ni-Co-based superalloys.As shown in η) [After high-temperature solution treatment, there were no second-phase particles in the matrix γ phase. The deformation of surface layer grains was less constrained due to the presence of free surfaces. The surface layer grains were easy to rotate to promote the movement of dislocations, thereby reducing the flow stress and hindering the occurrence of void nucleation in the surface layer grains. The rotation of the grains caused uneven surface deformation. The rotation of grains was considered as a possible explanation for surface roughening during plastic deformation ,50. The η) ,52 cause(1)Different grain sizes were controlled with high-temperature solution treatment, and the average grain size of the Ni-Co-based superalloy increased from 23.3 μm to 84.8 μm and the feature size decreased from 8.6 to 2.4 with increasing solution temperature.(2)t/d decreased from 8.6 to 2.4, the tensile strength σb decreased from 1221 MPa to 1090 MPa, the yield strength σs decreased from 793 MPa to 622 MPa, and the elongation decreased from 0.26 to 0.21 at room temperature. When t/d decreased from 8.6 to 2.4, σb decreased from 1458 MPa to 1132 MPa, σs decreased from 917 MPa to 730 MPa, and the elongation decreased from 0.31 to 0.28 at ultra-low temperatures.The flow stress decreased significantly with increasing grain size and increased significantly with decreasing deformation temperature. When (3)t/d and decreased with decreasing deformation temperature. Ultra-low temperatures enhanced the plastic deformation ability of the alloy. When t/d decreased from 8.6 to 2.4, the surface roughness of the specimen increased from 0.769 to 0.890 at room temperature and increased from 0.648 to 0.809 at ultra-low temperatures.The surface roughness of the specimen increased with decreasing (4)The coupled effects of surface roughening caused by free surface grains and hindered dislocation movement induced by grain boundaries during plastic deformation resulted in strain localization, causing fracture failure of the Ni-Co-based superalloys.This study carried out micro-tensile tests on Ni-Co-based superalloy thin sheets. The effects of grain size and deformation temperature on micro-tensile mechanical properties and microstructure evolution were analyzed, and the micro-tensile fracture mechanism was revealed. The main conclusions are as follows: